生物化学技术

Hammerhead ribozymes as typically used in their trans-cleaving form are defined by their hybridizing arms, the remainder of the molecule being essentially constant. A major determinant of the nature of the hybridizing arms is their sequence, which is in turn determined by the sequence of the R ...

The hammerhead ribozyme motif can be engineered to function as a sequence-specific endoribonuclease for use as a specific inhibitor of gene expression (1,2). However, efficient inhibition often requires high ribozyme concentrations and a large excess of ribozyme over substrate (3). ...

The ability to catalyze selected chemical reactions could be of great theoretical and practical value. Uhlenbeck first demonstrated a general method to design catalysts that could distinguish the intended substrates from other, very similar molecules (1). The reaction catalyzed w ...

Ribozymes first bind their target via complementary sequences like a conventional antisense strand and, subsequently, catalyze the hydrolyses of the cleavable motif. In the case of hammerhead ribozymes, this is a specific base triplet. Even though it has been described that there are 12 co ...

For naturally occurring antisense-regulated systems, it has been found that the rate for the association of complementary RNA strands in vitro reflects the biological effectiveness of the antisense RNA in vivo (1). Recently, a similar correlation has been identified for artificial HIV ...

In vitro selection methods are powerful tools for the selection of molecules with defined characteristics from complex starting populations (1,2). We have developed a powerful in vitro selection method for analysis of the hairpin ribozyme (3–5). The selection method relies on two sequen ...

There are several methods available for the direct and indirect detection of hammerhead ribozyme-mediated cleavage of substrate RNAs in vivo. The indirect methods include the use of antisense and/or mutant ribozyme constructs to differentiate between inactivation by cleavage a ...

Among the multiple publications that describe the use of ribozymes to knock out gene expression, only a few have directly proven that the ribozymes were catalytically active in vivo (1–3). Most studies rely on indirect evidence, such as a decrease in the level of the target RNA, and the comparison of the r ...

Trans-cleaving ribozymes can be targeted to cut specific RNAs in vitro, which has led to much interest in their potential to destroy specific messages inside cells. Here, we will describe a different reaction catalyzed by ribozymes that may allow them to be employed to “revise” genetic instruc ...

Ribozymes show promise as therapeutic agents for the downregulation of specific target RNAs inside cells. To demonstrate the efficacy of a given ribozyme treatment, one needs to show that the treatment ameliorates the disease condition relative to a control treatment. As part of that demo ...

Ribozymes are capable of catalyzing a variety of RNA cleavage, ligation, and splicing reactions. Of these three reaction types, ligation is the least-studied, primarily because of practical difficulties in achieving reasonable reaction efficiencies. Ribozyme-catalyzed lig ...

The development of in vitro transcription methods has facilitated research in RNA structural biology in recent years. There is now increasing interest in determining atomic resolution structures of RNA by X-ray crystallography. The first step in this process is the production of a homog ...

This chapter describes methods for the successful two-dimensional modeling of a small catalytic RNA. Our laboratory has used these methods to model and name the hairpin ribozyme family based on secondary structure predicted from computer modeling and mutagenesis (see Chapter 19) (1,2 ...

Successful crystallization of ribozymes and ribozyme domains depends on covalent homogeneity of the sample, conformational homogeneity of the preparation, and an efficient and broad sampling of solution conditions where crystals might nucleate and grow. Chapter 38 presents m ...

With the discovery of RNA catalysts and the role of RNA in many essential biological processes, our need to learn the fundamentals of RNA structure has become acute. X-ray crystallography is the only means available to determine the three-dimensional structure of many biologically intere ...

Artificial antisense RNAs and recombinant, trans-acting ribozymes have been developed whose ability to inhibit gene expression varies from negligible to profound. Although we are only beginning to understand the underlying reasons for this extreme variation, we can identify a num ...

Solid-support chemical synthesis of RNA (1), though costly, has the advantage of allowing the incorporation without restriction of any desired nucleotide sequence, including sequences which contain special modified nucleotides. For example, the crystal structure of an all-RNA h ...

The delivery and expression of hammerhead ribozymes to eukaryotic cells have largely been achieved using RNA polymerase II (pol II)-based promoters for transcription of ribozyme sequences. These constructs produce transcripts that are capped (5′-m7G) at the 5′-end and contain long tr ...

The hammerhead and hairpin ribozyme motifs were first identified and characterized in small, circular RNA molecules (satellite RNAs, viroids) associated with some diseases in plants (1). The ribozyme activity is necessary for replication of these RNAs, functioning to cleave linear c ...

To examine the effects of a ribozyme in VIVO, a major obstacle to overcome is the delivery of these molecules into the cells. A serious drawback of exogenous delivery is that the inhibitory effects of the nucleic acids so delivered are transient and require repeated administrations. Despite this, ...